This proposal seeks to characterize the structural dynamics of the bacterial multidrug efflux pump EmrD. The phenomenon of multidrug resistance is an increasingly significant obstacle to the clinical treatment of bacterial infections. One key strategy bacteria use to resist treatment with antibiotics is the expression of multidrug efflux pumps in the inner membrane. Drug/H+ antiporters (DHA), part of the major facilitator superfamily (MFS) of secondary transporters, couple the efflux of a variety of chemically dissimilar drug molecules out of the cell to the dissipation of an electrochemical gradient of protons across the membrane. Biochemical studies of two representatives of the DHA family, LmrP from Lactococcus lactis and MdfA from Escherichia coli, have shed light on the substrate specificity and the transport mechanism. A crystal structure of a related transporter, EmrD from E. coli, revealed a global fold similar to other MFS transporters but an unexpected conformation. The studies proposed here will utilize the biochemical data on LmrP and MdfA and the structural data on EmrD for the development of a dynamic, mechanistic model of drug/H+ antiport by this clinically important family of drug efflux pumps. Specific Aim 1 will evaluate the structure of the cytoplasmic gate of apo or substrate-bound EmrD to determine whether the crystal structure of EmrD represents a native state. Specific Aim 2 will characterize the conformations of the cytoplasmic gate of EmrD associated with proton binding and membrane energization. Specific Aim 3 will map substrate binding to the cytoplasmic surface of EmrD. The strategy of this investigation utilizes site-directed spin labeling, electron paramagnetic resonance (EPR) spectroscopy, and fluorescence spectroscopy to report on the structure of EmrD under various conditions. Structural data from EPR spectroscopy will provide information on the native state of EmrD in liposomes without detergents or crystal lattice forces. Additionally, these experiments will develop novel methodology to monitor the structural dynamics of a membrane protein under the influence of a stable proton gradient.